Device and method using integrated neuronal cells and an electronic device

ABSTRACT

The present invention provides a device of integrated neuronal cells interfaced with an electronic device and a method of producing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/194,693, filed Aug. 20, 2008, which is a continuation of U.S.application Ser. No. 10/496,476 (now U.S. Pat. No. 7,429,267), filedNov. 17, 2004, which is a National Stage application of PCT/US02/38670,filed Dec. 4, 2002, which in turn claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Applications Ser. Nos. 60/336,975, filedDec. 4, 2001 and 60/386,982, filed Jun. 6, 2002, all of whichapplications are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention is supported in part by funds from the U.S. Government(National Institutes of Health Grant Number AG012527), and the U.S.Government therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

The human brain is an exceedingly complex processing system, whichintegrates continual streams of incoming sensory input data with storedmemories, uses the input data and memories in complex decision processesat both conscious and unconscious levels, and on the basis of theseprocesses generates observable behaviors by activation of its motor ormovement control pathways and the muscles which these innervate.

In the United States, approximately 12,000 people each year suffer someform of spinal cord injury (SCI), with over 275,000 people chronicallyparalyzed from SCI. There are two general types of SCI: complete andincomplete lesions. Complete lesions leave the patient with no motor,sensory, or autonomic function below the level of the lesion.Transection of the spinal cord is the most obvious cause of a completelesion. The level of the injury in the spinal cord determines exactlywhat function will be lost, as the spinal nerves that exit the cordbelow this are absolutely unable to transmit signals to or from thebrain. Incomplete lesions can take a variety of forms, and depending onthe nature of the trauma, a range of motor and sensory abilities may bepresent.

Non-traumatic pathologies such as stroke and Parkinson's disease arealso often characterized by a patient's inability to successfullytranslate a desire into the appropriate motions of the relevant limbs.Central nervous system pathologies are often responsible for varyinglevels of paralysis, which cause immense suffering in the affectedpopulation.

Rehabilitation efforts for these patients usually focus on teachingmeans for using still-functioning limbs to carry out desired tasks,while trying, when possible, to recover some function in the affectedlimbs. In addition, a range of technologically advanced and expensivedevices have been built and tested on patients with limited success.Amongst these are muscle-stimulation devices, which include electrodesthat are mounted on a patient's muscles in a paralyzed limb. In responseto a command, the electrodes drive current into the muscles, causing thecontraction thereof. The resultant motion of the limb is typicallyrough, and the unnatural stimulation protocols often leave the patient'smuscles tired, even after performing only a small number of tasks.

U.S. Pat. Nos. 5,178,161; 5,314,495 and 4,632,116 provide the use ofmicroelectrodes to interface between control electronics and humannerves.

U.S. Pat. No. 4,649,936 discloses an electrode cuff for placement arounda nerve trunk, for generation of unidirectional propagating actionpotentials.

U.S. Pat. No. 4,019,518 provides methods for using an electricalstimulation system to selectively stimulate portions of the body.

U.S. Pat. Nos. 5,776,171; 5,954,758 and 6,026,328 disclose methods anddevices for stimulating muscles of limbs of the body, so as to achievemotion and control of the limbs in patients with central nervous systemdisabilities. Limb motions in each limb are commanded by external meansand communicated via radio waves to an apparatus implanted in the limb.Actual motion of the limb is monitored and compared to the commandedmotion with the goal of attaining real-time control of the limb.

U.S. Pat. No. 5,748,845 provides a device for controlling limbs ofpatients with central nervous system disabilities. The activity of ahealthy muscle is sensed, analyzed, and used to determine inputparameters to a control system of the device. Both external mechanicalapparatus and direct electrical stimulation of muscle tissue aredescribed as means for inducing movement of the disabled limb.

Other methods and devices for sensing muscular contractions and forapplying muscular stimulation are provided by U.S. Pat. Nos. 6,091,977;6,104,960; 6,086,525; 4,926,865; 4,392,496 and 6,146,335.

U.S. Pat. No. 6,119,516 discloses a biofeedback system, optionallyincluding a piezoelectric element, which measures the motions of jointsin the body. U.S. Pat. No. 5,069,680 provides the use of a piezoelectriccrystal as a muscle activity sensor. U.S. Pat. Nos. 4,602,624 and5,505,201 disclose techniques for making implantable electrodes. U.S.Patent Application No. 20020161415 further provides a plurality ofelectrodes, which are adapted to be placed in a vicinity of a motornerve that innervates the skeletal muscle.

Despite these advances there remains a need for a means to communicatewith the regions of the brain via an external interface which isplastically adaptive, sensitive, and responsive to the subtleties ofnerve transmission.

SUMMARY OF THE INVENTION

The present invention provides integrated neuronal cells, which may bemechanically elongated to lengths of greater than one centimeter,interfaced with an electron device. Said neuronal cells are capable oftransmitting electrical signals to and receiving electrical signals froman electronic device and may comprise nerve bundles.

The present invention further provides a method of producing integratedneuronal cells interfaced with an electronic device. The method providesplacing a first neuronal cell on an electronic device which is adjacentto a membrane which contains a second neuronal cell and allowing thecells to integrate. In a preferred embodiment, the integrated neuronalcells are elongated using a mechanical device.

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of integrated elongated neuronalcells attached to an implantable membrane and an electronic device.

FIG. 2A and 2B show integrated elongated neurons attached to amulti-electrode array.

DETAILED DESCRIPTION OF THE INVENTION

The primary functional constituents of the spinal cord are myelinatedaxons and neurons. Signals travel from brain to body and back via theseaxons which synapse to spinal neurons communicating with the targetedbody region. At present, electronic devices to interface with thiscomplex system of communication include wires or devices placed inproximity with the region of the brain of interest. However, only verycrude signals can be recorded from or transferred to the brain in thisfashion.

An improved device has now been found to bridge electronic devices withbiological processes. The device is integrated neuronal cells physicallyattached to or grown onto an implantable membrane and an electronicdevice (FIG. 1). The electronic device is used to send signals to orreceive signals from the brain and/or other peripheral regions of thebody. The advantage of the current device is that it comprises a living,spontaneously adaptive system of neuronal cells and an electronicdevice. Furthermore, when the neuronal cells have been mechanicallyelongated they allow for the electronic device to be located eitheroutside of the body or implanted at an internal location which can betolerated by the patient.

One aspect of the invention provides a method of producing integratedneuronal cells interfaced with an electronic device. The methodcomprises placing a first population of neuronal cells on an electronicdevice which is adjacent to a membrane containing a second population ofneuronal cells. The membrane may be any material, however a biologicallyabsorbable material is preferable as it is more compatible fortransplantation into tissue. The two populations of neurons are allowedto mature and integrate among each other, including growth of axonsacross the border between the electronic device and membrane. In apreferred embodiment, the electronic device and membrane areprogressively separated using, for example, a micro-stepper motor system(see, e. g. , U.S. Pat. No. 6,264,944, herein incorporated in itsentirety), resulting in two populations of cell bodies connectedtogether by elongated fascicular axon tracts.

Using the method of the invention, rat dorsal root ganglion wereattached to a membrane and a multi-electrode array electronic device(FIGS. 2A and 2B). It was found that dorsal root ganglion cells could beelongated to lengths of over three to five centimeters at an averagerate of six millimeters per day. The maximal rate obtained was eightmm/day sustained for 24 hours with no evidence of axonal breakage ordamage. Modified elongation devices may be engineered to produceneuronal cells elongated to lengths of 15 cm.

Short axon segments were found to be very sensitive to the rate ofstretch-growth. If axons were allowed to grow at a slow to moderate ratefor several days, the axons could be ramped to higher stretch rateswithout axonal damage. Axons that were grown for one day at a rate of 1mm/day and then ramped to 6 mm/day showed a significant amount of axonalbreakage after 2 days. However, if the axons were allowed to elongatefor a period of 3 days at 1 mm/day and then ramped to 6 mm/day, therewas no evidence of axonal breakage.

Adult and embryonic DRG cells were elongated. Adult DRG cells tooklonger to initiate regenerative growth in culture following dissectionand dissociation.

Approximately one additional week in culture (compared to embryoniccells) was required for adult DRGs to grow sufficient axons across theelongation interface. Adult DRG cells also were constrained to a slowerrate of elongation; a maximum of 1 mm/day for 7 days was attainable.

Immunocytochemistry of the elongated fascicular axon tracts revealed anormal cytoskeleton containing phosphorylated neurofilament, tau andβ-tubulin protein expression. The results indicated that the heavyneurofilament (NF-H, 200 kDa) was present throughout the entireelongated axon length. All axons studied showed the same result overseveral studies and at each rate and length. Antibodies to thephosphorylated NF-H (SMI-32) and a non-specific NF-200 antibody hadsignificant reactivity in all elongated axons. β-tubulin was alsoidentified using the SMI-61 and SMI-62 antibodies for unassembled andassembled-tubulin, respectively. Moreover, tau protein expression, whichis reportedly the slowest of the transported cytoskeletal proteins, wasfound in significant amounts throughout each and every elongated axon.Similar results were found for adult DRG elongated axons.

Transmission electron micrographs were prepared for the cross-sectionsof both stretch-induced elongation and growth cone induced growth of DRGaxons. Microtubules were counted and cross-sectional areas weremeasured. The results showed that stretch induced axon elongation leadsto a hypertrophy of axonal caliber. On average, the axon cross-sectionalarea increased by 30% and the median cross sectional area increased byalmost 50%.

In a preferred embodiment of the present invention, neuronal cells arederived from any cell that is a neuronal cell (e. g., cortical neuronsor dorsal root ganglion neurons) or is capable of differentiating into aneuronal cell (e, g., stem cell) and can function in the central nervoussystem or peripheral nervous system. Moreover, these cells may bederived from cell lines or other mammalian sources such as donors orvolunteers. Furthermore, the neuronal cells may be singular, integratedneuronal cells or a plurality of integrated neuronal cells (i.e., anintegrated nerve bundle) interfaced with an electronic device. When theelectronic device is interfaced with a nerve bundle, a signal may betransmitted from an external control module to stimulate one, some, orall of the axons in the nerve bundle, thereby causing, for example,contraction of a muscle. Preferably, the control module containscircuitry which regulates the magnitude, frequency, and/or duration ofthe electric signal transmitted by the electronic device.

In another preferred embodiment the electronic device interfaced with aneuronal cell or nerve bundle comprises a multi-electrode array, asexemplified herein, or any electronic device capable of transmitting andreceiving electrical signals including, but not limited to, anelectrode, microchip or sensor/actuator.

It is contemplated that the device of the invention will be useful as asource of transplant material for patients with spinal cord injury aswell as other nerve lesions such as those derived from aneurodegenerative disease. The device may also be used to restorecommunications of a severed limb or organ of the peripheral nervoussystem with the central nervous system. Methods for transplantation ofthe cells of the device of the invention are well-known to those ofskill in the art of cell transplantation. Suitable transplant materialmay be evaluated by using well-known electrophysiological andfluorescence techniques to demonstrate that digital signals can be sentfrom the electronic device to the attached neurons and stimulate anaction potential to be received by the distant neurons. Likewise,distant neurons grown on the transplantable membrane may be stimulatedto demonstrate that a signal can be received by the electronic device.

Transplanted cells are oriented such that the distant neuron isimplanted in the brain or at or near a site of nerve damage and theelectronic device is exteriorized. In this manner, the signals generatedby the brain cells or viable cells at or near the site of nerve damagemay be exteriorized. Signals received from the brain, for example, maybe used to control external prostheses, such as an assist robot or anartificial arm; computers or computer displays; or functional electricalstimulation of muscles of paralyzed individuals for the restoration orenhancement of movement.

As one of skill in the art can appreciate, the device of the presentinvention would permit a signal to be sent from the brain to theelectronic device to allow a subject to control a device such as aprosthesis or robot. That is, the subject would transmit a signal, theimpulse would be transmitted by an implanted neuronal cell interfacedwith an electronic device, an external control module would measure andconvert the impulse received by the electronic device from the brain,the external control module would then transmit a signal to a secondelectronic device interfaced with a prosthesis or robot which undertakesthe activity that is imagined or intended. Known methods for measuringbrain electrical impulses are described, for example, in U.S. Pat. No.4,862,359. Methods of controlling robotics or prostheses are disclosedin U.S. Pat. No. 6,171,239 and Amirikian, et al. ((1999) Can. J. Exp.Psych. 53: 21-34.

Furthermore, the device of the present invention would permit a signalto be sent to the brain from the electronic device. For example, aphototransistor microprocessor array may serve as the electronic deviceattached to the integrated neuronal cells. The distant neuronal cellsattached to an implantable membrane are then transplanted into thelateral geniculate nucleus (LGN) region of the thalamus, which functionsas a junction box for visual signals. Elongation of the neuronal cellsallows the phototransistor microprocessor array to be exteriorized toreceive light impulses outside the cranium. These impulses are then sentdeep into the brain by the distant neurons. Behavioral tests andelectrophysiologic analysis are performed to evaluate the function ofthe implant.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Embryonic Dorsal Root Ganglion Cell Isolation

Dorsal root ganglia (DRG) were isolated from E15 (EO is the day aftermating) rat embryos. Dissected ganglia were held in Lebovitz L-15 mediumduring the isolation. Dissociated cultures were treated with 0.25%trypsin (Sigma-Aldrich, St. Louis, Mo.) in a cell dissociation buffer(INVITROGENTM, Carlsbad, Calif.) and incubated at 37° C. for 45 minutes.Trypsin activity was stopped with the addition of L-15 medium+20% fetalbovine serum (FBS; Hyclone, Logan, Utah) and cells were centrifuged at1000 rpm for five minutes. After discarding the supernatant, the cellpellet was resuspended in 2 mL of complete medium consisting of NeuralBasal Medium (INVITROGEN™, Carlsbad, Calif.) supplemented with B27(INVITROGEN™, Carlsbad, Calif.), 1% FBS (Hyclone, Logan, Utah), 1 mML-Glutamine (INVITROGEN™, Carlsbad, Calif.), and 2.5 g/L glucose. TheDRGs were then triturated ten times with a fire-polished pipette. Cellswere counted and plated on a collagen-or laminin-coated surface at adensity of 1-2×10⁶ cells per mL.

Whole DRGs were plated immediately following isolation in completemedia. Plating of whole DRGs allows for very high plating densities andleads to much larger axon fascicles during mechanical elongation. Thewhole DRGs may be “softened” by a one minute trypsin treatment followedby trypsin inactivation with L-15+20% FBS. Pelleted ganglia areresuspended in complete media gently using a Pasteur pipette andimmediately plated.

EXAMPLE 2 Adult Dorsal Root Ganglion Cell Isolation

The adult DRGs were dissected from adult Sprague-Dawley rats of at leasteight weeks of age (Scott (1977) J. Neurobiol. 8 (5): 417-27). Briefly,the spinal column was removed from the brain-cervical junction to belowL1. Attached tissue and the spinous processes were removed using a pairof roungers. The column was cut with a pair of tough iris scissorsstarting at the cervical end, cutting through the dorsal roof bone(staying centered as not to damage the ganglia). The column was thenseparated lengthwise in half by carefully cutting through the cord andventral side of the column. The spinal cord and menengies along theinner spinal column were removed using fine forceps and the DRGS werepulled from the foramen. Using small spring scissors, the nerves on bothsides of the DRG were cut. Two incisions were made on each side ofganglia and placed into bovine serum albumin (BSA) coated tubescontaining L-15 medium during the isolation.

Adult DRGs are myelinated and must be dissociated to separate theneurons from the surrounding tissues and supporting cells. DissectedDRGs were treated in 0.25% collegenase-P (Boehringer Mannheim, Germany)in Neural Basal medium for 1.5 hours. All the DRGs and tissues werepelleted and resuspended in 0.25% trypsin in cell dissociation bufferfor an additional 1.5 hours. Trypsin activity was stopped with 20% FBSin Neural Basal medium and the cells were centrifuged. The pellet wasresuspended in complete medium and mechanically separated using afire-polished Pasteur pipette until the DRGs were completelydissociated.

An almost pure suspension of DRGs was obtained by passing thedissociated product through a BSA gradient. A two-layer BSA gradient (5%and 10%) was prepared by adding 5 mL of a 5% BSA solution in a 15 mLcentrifuge tube. Using a Pasteur pipette, the second layer was addedbelow the 5% BSA by slowly pipetting the 10% BSA solution with thePasteur pipette tip at the bottom of the tube. The 5% BSA floats on topthe 10% BSA. The dissociated DRG suspension was carefully placed on topof each gradient by adding it drop by drop along the side of the tube.The gradient tubes were centrifuged at 100×g for seven minutes. Most ofthe DRG cells and non-neuronal cells pelleted while the myelin andSchwann cells were found in the upper fractions of the gradient. Pelletswere resuspended in 5 mL of complete media, and carefully placed on asecond BSA gradient prepared as described. The gradients werecentrifuged at approximately 90×g for no more than five minutes. Thiscentrifugation step removed most of the smaller non-neuronal cells fromthe larger DRG cells. The pellets were resuspended in complete medium,counted and plated.

EXAMPLE 3 Axon Elongation Device

Dorsal root ganglion cell axons were grown by tension induced elongation(Smith, et al. (2001) Tissue Eng. 7: 131-138). The device consistentlyseparates two adjoining substrates on which neural cells are cultured.The adjoining substrates were placed such that axons growing in culturecould grow across the interface between the two substrates easily. Thebottom substrate, an electronic device, was placed in the bottom of theelongation device on which a stationary population of neurons wascultured. An overlapping ACLAR® substrate (Honeywell, Berkshire, UK), i.e., the towing substrate, was placed on top of the electronic devicesubstrate and served as the moving population of cells. Once the neuronsand their axons matured and synapsed across the bottom and towingsubstrate interface, the two substrates were separated using amicro-stepper motor system (Smith, et al, (2001) Tissue Eng. 7: 131-138;U.S. Pat. No. 6,264,944). The result was two populations of cell bodiesconnected together via elongated fascicular axon tracts.

EXAMPLE 4 Elongation Device Preparation, Plating, and Maintenance

Cell culture plates, the ACLAR® surface, and the electronic device werecoated with collagen prior to DRG plating. After ACLAR® was cut intodesired sizes, it was washed with laboratory soap and rinsed well.ACLAR® was then treated in 1 M NaOH for 24-48 hours, rinsed well insterile water, then bathed in 100% ethanol for 10 minutes. The ACLAR®was then allowed to dry on a sterile rack in a cell culture hood. TheACLAR® substrate was attached to the elongation device framework usingmedical grade RTV silicone (NuSil, Carpinteria, Calif.). During curing,RTV silicone releases acetic acid, which can be lethal to cells,therefore the silicone was allowed a minimum of three days to completelycure prior to plating cells. Twenty-four hours after assembly, theACLAR® surface was treated with 10 μg/mL poly-L-lysine (PLL) for 4hours. The PLL solution was removed and the ACLAR® was allowed to dryfor one hour and the surface was subsequently rinsed three times withsterile water. After the silicone has completely cured the ACLAROsurface was coated with type 1 rat-tail collagen (Becton Dickinson,Franklin Lakes, N.J.). Collagen was spread over the surface (10-20 μLper cm²) and polymerized by exposure to ammonia vapors for two minutes.The collagen was then allowed to dry completely in a cell culture hoodbefore plating cells. The electronic device was sterilized and coatedwith collagen in a similar manner.

The hydrophobic collagen surface provides that cells can be plated inany desired arrangement by applying the cell suspension in a puddle andallowing the cells to attach for about 2-4 hours before the chamber isflooded with media. Embryonic, dissociated cells cultures were plated ata density of 1-2×10⁶ cell per mL of which 500 μL was plated along theadjoining substrate interface of the elongation device. Whole embryonicDRGs were plated from 6-8 pups in 500 μL of complete medium along thesubstrate interface of the elongation device. Dissociated DRG cells fromone adult rat were resuspended in 1 mL of complete medium of which 500μL was plated on each of two elongation devices.

After cell attachment, the elongation device chamber was flooded withcomplete medium with mitotic inhibitors, 10 μM FdU and 10 μM Uridine.The media was changed every two to three days and the mitotic inhibitorsapplied once a week. On day five, the elongation device was turned onand left undisturbed without changing the medium. For long-termexperiments, the media was changed once a week.

EXAMPLE 5 Axon Elongation Scheme

Five days after plating, the DRG cells were elongated. Elongation wascontrolled by displacement of the towing membrane. Since stretch inducedgrowth was initially strain limited, elongation began at a slow rate andwas increased to the desired growth rate. Stretch rate was programmedinto the motion control device by choosing a displacement, and a restingtime in a step-wise fashion. For example, 1 mm/day was programmed as 1μm displacements every 86.4 seconds.

Elongation started at 1 mm/day (1 μm every 86.4 seconds) for the first24 hours. The elongation rate was then increased by 1 mm/day every 6 to12 hours until the maximum elongation rate was achieved. In the case ofa maximal rate of 8 mm/day, the ramping was slower and at 12 to 24 hourintervals to allow the axons to increase in length.

EXAMPLE 6 Tissue Fixation, Immunocytochemisty and Electron Microscopy

For immunocytochemistry, the elongated tissue was fixed with 4%paraformaldehyde for 60 minutes. Following three rinses inphosphate-buffered saline (PBS), elongated axons were blocked with 4%Normal Goat Serum (NGS) in PBS at room temperature for 60 minutes. Theprimary and secondary antibodies were applied in 4% NGS, 0.1% Triton Xin PBS for 60 minutes each. Primary antibodies targeted to the 200 kDaneurofilament fragment where NF200, diluted 1:400 (Sigma-Aldrich, St.Louis, Mo.), and SMI-32, diluted 1:400 (Sternberger Monoclonals,Lutherville, Md.). Antibodies to β-tubulin where SMI-61 & 62, diluted1:400 (Sternberger Monoclonals, Lutherville, Md.) and the antibody toTau was diluted 1:400 (Dako, Denmark). Fluorescent secondary antibodieswere used according to manufacturer's instructions (Molecular Probes,Eugene, Oreg.).

For transmission electron microscopy, elongated axons were fixed in 4%paraformaldehyde, 2% glutaraldehyde, in 0.1 M sodium cacodylate buffer,overnight at 4° C. To prevent any damage to the axons during theirremoval from the elongation device, the tissue was supported in 2% agar.Melted agar was allowed to cool to approximately 45° C. then gentlypipetted over the tissue. The agar was allowed to cool and harden at 2°C. With a #11 scalpel, the substrates were carefiilly cut loose and thetissue removed. The tissue was then washed in the same buffer andpost-fixed in 1% osmium tetroxide for 1 hour at 4° C. After anotherbuffer wash, the sample was dehydrated in a graded ethanol series beforeinfiltration and embedding in epoxy resin (EMbed-812; ElectronMicroscopy Sciences, Fort Washington, Pa.). Thin sections were cut at800 Å and placed on fonnvar-coated grids. After staining with uranylacetate and lead citrate, sections were examined with a JEOL 100CXtransmission electron microscope.

For scanning electron microscopy, elongated axons were fixed in 4%paraformaldehyde, 2% glutaraldehyde, in 0.1 M sodium cacodylate buffer,overnight at 4° C. The sample was rinsed and post-fixed in 1% osmiumtetroxide for 30 minutes. After another buffer wash, the sample wasdehydrated in a graded ethanol series followed by a drying step of twoapplications of Hexamethyldisilazane (HMDS, Electron MicroscopySciences, Fort Washington, Pa.) of ten minutes each.

1. A method of producing a composition of neuronal cells interfaced withan electronic device comprising: (a) providing a first isolated neuronalcell interfaced with the electronic device; and (b) providing a secondisolated neuronal cell; wherein said first isolated neuronal cell isintegrated to the second isolated neuronal cell via elongated axonsstretched to greater than 1 cm in length by stretching the axons at aslow stretch rate of about 1 mm/day followed by stretching the axons ata higher stretch rate of at least 2 mm/day to obtain axons elongated togreater than 1 cm in length.
 2. The method of claim 1, wherein saidaxons are elongated for about 3 days at the slow stretch rate.
 3. Themethod of claim 1, wherein said higher stretch rate is about 6 mm/day.4. The method of claim 1, wherein said axons are elongated at the higherstretch rate until the axons are greater than 1 cm in length.
 5. Themethod of claim 1, wherein said axons are greater than 3 cm in length.6. The method of claim 1, wherein the neuronal cells transmit anelectrical signal to the electronic device.
 7. The method of claim 1,wherein the neuronal cells receive an electronic signal from theelectronic device.
 8. The method of claim 1, wherein the integratedneuronal cells comprise fascicular axon tracts.
 9. The method of claim1, wherein the axons are stretched using a microstepper motor system.10. The method of claim 1, wherein the isolated neuronal cells areattached to an implantable membrane.
 11. The method of claim 1, whereinthe electronic device is a multi-electrode array.
 12. A method ofproducing a composition of neuronal cells interfaced with an electronicdevice comprising: (a) providing a first isolated neuronal cellinterfaced with the electronic device; and (b) providing a secondisolated neuronal cell; wherein said first isolated neuronal cell isintegrated to the second isolated neuronal cell via elongated axonsstretched to greater than 1 cm in length by stretching the axons at aslow stretch rate of about 1 mm/day for about 3 days followed bystretching the axons at a higher stretch rate of about 6 mm/day for aperiod of time sufficient to obtain axons elongated to greater than 1 cmin length.